Toxicology studies of substances have traditionally relied on unicellular organisms (for example, the Ames test or the yeast carcinogenic assay described in U.S. Pat. No. 4,997,757) or in vitro systems for toxicity testing and the prediction of human risk. However, there are many factors that make it difficult to extrapolate from such data to human risk including cellular affinity of the substance, uptake and distribution differences between single cells and whole animals, metabolism of the substance, and cascade effects where the effect of the substance is mediated through a cellular process. These same factors can affect the progress of pharmaceutical research and development as well when attempting to determining and/or predicting the effects of an analyte in an animal system.
Further, the end-point of traditional animal based toxicology studies is typically determination of an LD50 (the dose at which 50% of the test animals die). Dead animals may be subjected to further analysis, for example, histopathology, but such analysis is generally labor intensive and relatively insensitive. MacGregor, et al (Fundamental and Applied Toxicology, 26:156-173, 1995) have reviewed molecular end-points and methods of routine toxicity testing including the following: damage-inducible genes in individual cells; bacterial models of toxicity; screening of stress-gene expression using hybridization or polymerase chain reaction; hybridization probes for detection of chromosomal aberrations; single cell electrophoresis assays; and in vivo animal studies involving animal sacrifice and subsequent analysis of tissue/cellular damage.
P450 enzymes have been shown to be involved in the biosynthesis of steroids and cholesterols and in metabolizing drugs or xenobiotics. P450 enzyme induction is a result of fluctuations in levels of steroids and cholesterols, or of repeated exposure to drugs or xenobiotics. Changes in P450 enzyme levels result in changes in plasma and/or tissue levels of the drugs they metabolize, which in turn affects the stability, efficacy and toxicity of those drugs. Among P450 superfamilies, the Cyp3A family typically accounts for 14-31% of total P450 present in human liver microsomes and for 50-60% of the drug metabolic activity. (Toide et al. (1997) Arch. Biochem. and Biophysics 338:43-49). Clones encoding distinct Cyp3A forms have been isolated from human, rat, guinea pig and mice, including Cyp3A11 in mice and CYP3A4 in humans. Therefore, P450 enzyme expression, particularly the Cyp3A family of genes, is a vital pharmacological parameter of bioavailability of pharmaceutical agents, as well as of drug-to-drug interactions.
Currently, conventional assays for P450 gene regulation are laborious and time-consuming, for example Northern blots, Western blots, RT-PCR or reporter assays ex vivo. In addition, expression of P450 genes in cell line has proven difficult. Thus, there remains a need to directly monitor P450 gene regulation in real-time in live animals.